The proteins eluted with boiled SDS-PAGE sample buffer were separated by SDS-PAGE.
Briefly, unheated samples in Laemilli buffer were separated by SDS/PAGE, blotted onto PVDF membranes and then blocked in 5% non-fat milk.
Proteins suspended in Laemmli buffer were separated by SDS-PAGE (10% gels), transferred onto PVDF membranes (Millipore, US) and revealed with specific antibodies.
For immunoblot experiments, 20 to 50 μg of lysates in SDS loading buffer were separated by SDS-PAGE.
Denatured protein samples with 1 × loading buffer were separated by 12%% polyacrylamide gel electrophoresis followed by transfer to a pure nitrocellulose blotting membrane (Pall Corporation).
Proteins extracted using RIPA buffer were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrotransfered and immobilized on a nitrocellulose membrane.
Cell lysates (20 μg protein) in denaturing buffer were separated by SDS electrophoresis (12% SDS polyacrylamide gels) and electroblotted onto nitrocellulose membranes at 12 V for 15 min in 20 mM CAPS/NaOH buffer pH 10.3 containing 10% (v/v) methanol and 0.1% (w/v) SDS.
Normalized protein lysates or virus pellets resuspended in Laemmli buffer were separated by SDS-PAGE, transferred to PVDF membranes, and probed for HCV-E2 (mAb 3/11) or HCV-Core (mAb C750, Thermo Scientific) and expression quantified by enhanced chemiluminescence and densitometric analysis using ImageJ software.
In order to avoid a reaction between the water fugacity buffer and the specimen, a double-capsule assemblage was designed such that the water fugacity buffer was separated from the specimen by an inner metal jacket permeable to hydrogen and impermeable to other components.
The labelled probe, dissolved in 50 µl of TE buffer, was separated from unincorporated nucleotides by passing through an Illustra Nick column (GE Healthcare, Buckinghamshire, UK) and heat denatured as described by the manufacturer.
